scholarly journals Separation of human myeloma cells from bone marrow aspirates in multiple myeloma and their proliferation and M-protein secretion in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 562-566 ◽  
Author(s):  
K Iwato ◽  
M Kawano ◽  
H Asaoku ◽  
O Tanabe ◽  
H Tanaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Secretion ◽  
Secretion Rate ◽  
M Protein ◽  
Cell Fraction ◽  
Bone Marrow Mononuclear Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cells

Abstract Human myeloma cells were purified from bone marrow aspirates from patients with multiple myeloma (MM) by Percoll discontinuous density- gradient centrifugation, E rosette formation and treatment with antimyelomonocytic antibody (Leu M1), plus complement. Thus, the purified cell fraction consisted of greater than 90% myeloma cells, even when as little as 15% myeloma cells were contained in bone marrow mononuclear cell fraction, determined by morphological and immunologic examinations. With highly purified myeloma cells from 29 patients with IgG type MM, biologic characteristics such as spontaneous proliferation (3H-TdR uptake) and M-protein secretion rate in vitro were evaluated. Both activities varied among patients within stage I and III, and a 3H- TdR uptake of 255–24, 132 cpm/4 x 10(4) cells, and an M-protein secretion rate of 9 to 72 pg/cell/day, respectively, were recorded. However, in each patient, there was no correlation between 3H-TdR uptake and M-protein secretion rate. These results thus suggest that 3H- TdR uptake and M-protein secretion rate of highly purified myeloma cells are independent biologic parameters, not associated with the clinical stages, and the purification of myeloma cells we describe can contribute to further studies on the biologic characteristics and to understanding of the pathophysiology involved in MM.

scholarly journals Separation of human myeloma cells from bone marrow aspirates in multiple myeloma and their proliferation and M-protein secretion in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 562-566 ◽  
Author(s):  
K Iwato ◽  
M Kawano ◽  
H Asaoku ◽  
O Tanabe ◽  
H Tanaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Secretion ◽  
Secretion Rate ◽  
M Protein ◽  
Cell Fraction ◽  
Bone Marrow Mononuclear Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cells

Human myeloma cells were purified from bone marrow aspirates from patients with multiple myeloma (MM) by Percoll discontinuous density- gradient centrifugation, E rosette formation and treatment with antimyelomonocytic antibody (Leu M1), plus complement. Thus, the purified cell fraction consisted of greater than 90% myeloma cells, even when as little as 15% myeloma cells were contained in bone marrow mononuclear cell fraction, determined by morphological and immunologic examinations. With highly purified myeloma cells from 29 patients with IgG type MM, biologic characteristics such as spontaneous proliferation (3H-TdR uptake) and M-protein secretion rate in vitro were evaluated. Both activities varied among patients within stage I and III, and a 3H- TdR uptake of 255–24, 132 cpm/4 x 10(4) cells, and an M-protein secretion rate of 9 to 72 pg/cell/day, respectively, were recorded. However, in each patient, there was no correlation between 3H-TdR uptake and M-protein secretion rate. These results thus suggest that 3H- TdR uptake and M-protein secretion rate of highly purified myeloma cells are independent biologic parameters, not associated with the clinical stages, and the purification of myeloma cells we describe can contribute to further studies on the biologic characteristics and to understanding of the pathophysiology involved in MM.

scholarly journals Identification of immature and mature myeloma cells in the bone marrow of human myelomas

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 564-570 ◽  
Author(s):  
MM Kawano ◽  
N Huang ◽  
H Harada ◽  
Y Harada ◽  
A Sakai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adhesion Molecules ◽  
Proliferative Activity ◽  
M Protein ◽  
Color Analysis ◽  
Myeloma Cells ◽  
A Cell ◽  
Immature Cells ◽  
Human Myeloma Cells

Abstract With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

scholarly journals Sensitive inhibitory effect of interferon-alpha on M-protein secretion of human myeloma cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1718-1722 ◽  
Author(s):  
H Tanaka ◽  
O Tanabe ◽  
K Iwato ◽  
H Asaoku ◽  
H Ishikawa ◽  
...  
Keyword(s):  
Interferon Alpha ◽  
Protein Secretion ◽  
Inhibitory Effect ◽  
M Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Proliferation ◽  
Myeloma Cells ◽  
Ifn Alpha ◽  
Human Myeloma Cells

Abstract The effects of interferon-alpha (IFN alpha) on in vitro proliferation and M-protein secretion in human myeloma cells were investigated. Human myeloma cells were purified from bone marrow aspirates in 12 multiple myeloma patients. Purified myeloma cells were cultured for 48 hours with IFN alpha at its lower concentrations (0.1 to 100 U/mL). The cells were then pulsed with 3H-TdR for the last 12 hours and 3H-TdR uptake was measured (in vitro proliferation). After 48-hour culture, supernatants were harvested and the amount of M-protein in these fluids were measured by enzyme-linked immunosorbent assay (ELISA) (in vitro M- protein secretion). In vitro M-protein secretions of myeloma cells were significantly suppressed even at 0.1 U/mL of IFN alpha, while 3H-TdR uptakes were not so suppressed until 10 or 100 U/mL of IFN alpha were added. The expressions of secretory immunoglobulin (Ig) mRNA of these myeloma cells were also selectively suppressed by IFN alpha. Furthermore, after IFN alpha had been administered intramuscularly, 3 to 6 x 10(6) U/d for at least 1 month, in vitro M-protein secretions of these myeloma cells were decreased compared with those before IFN alpha administration. Therefore, these results suggest that IFN alpha has more sensitive inhibitory effect on M-protein secretion of human myeloma cells rather than on myeloma cell proliferation.

scholarly journals Sensitive inhibitory effect of interferon-alpha on M-protein secretion of human myeloma cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1718-1722
Author(s):  
H Tanaka ◽  
O Tanabe ◽  
K Iwato ◽  
H Asaoku ◽  
H Ishikawa ◽  
...  
Keyword(s):  
Interferon Alpha ◽  
Protein Secretion ◽  
Inhibitory Effect ◽  
M Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Proliferation ◽  
Myeloma Cells ◽  
Ifn Alpha ◽  
Human Myeloma Cells

The effects of interferon-alpha (IFN alpha) on in vitro proliferation and M-protein secretion in human myeloma cells were investigated. Human myeloma cells were purified from bone marrow aspirates in 12 multiple myeloma patients. Purified myeloma cells were cultured for 48 hours with IFN alpha at its lower concentrations (0.1 to 100 U/mL). The cells were then pulsed with 3H-TdR for the last 12 hours and 3H-TdR uptake was measured (in vitro proliferation). After 48-hour culture, supernatants were harvested and the amount of M-protein in these fluids were measured by enzyme-linked immunosorbent assay (ELISA) (in vitro M- protein secretion). In vitro M-protein secretions of myeloma cells were significantly suppressed even at 0.1 U/mL of IFN alpha, while 3H-TdR uptakes were not so suppressed until 10 or 100 U/mL of IFN alpha were added. The expressions of secretory immunoglobulin (Ig) mRNA of these myeloma cells were also selectively suppressed by IFN alpha. Furthermore, after IFN alpha had been administered intramuscularly, 3 to 6 x 10(6) U/d for at least 1 month, in vitro M-protein secretions of these myeloma cells were decreased compared with those before IFN alpha administration. Therefore, these results suggest that IFN alpha has more sensitive inhibitory effect on M-protein secretion of human myeloma cells rather than on myeloma cell proliferation.

scholarly journals Identification of immature and mature myeloma cells in the bone marrow of human myelomas

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 564-570 ◽  
Author(s):  
MM Kawano ◽  
N Huang ◽  
H Harada ◽  
Y Harada ◽  
A Sakai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adhesion Molecules ◽  
Proliferative Activity ◽  
M Protein ◽  
Color Analysis ◽  
Myeloma Cells ◽  
A Cell ◽  
Immature Cells ◽  
Human Myeloma Cells

With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

Angiopoietin-1 and Osteopontin Expression by CD138+ Myeloma Cells Rather Than VEGF and CD45 Correlates with Bone Marrow Angiogenesis in Multiple Myeloma Patients at the Diagnosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2501-2501
Author(s):  
Nicola Giuliani ◽  
Simona Colla ◽  
Francesca Morandi ◽  
Sabrina Bonomini ◽  
Mirca Lazzaretti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Linear Trend ◽  
The Other ◽  
Vegf Mrna ◽  
Myeloma Cells ◽  
Other Hand ◽  
Significant Difference ◽  
Angiopoietin 1

Abstract Bone marrow (BM) angiogenesis is increased in Multiple Myeloma (MM) patients and correlates with disease progression and patient survival. Myeloma cells secrete the main endothelial growth factor VEGF. In mouse models VEGF secretion as well as the angiogenic properties of MM cells correlate with the lack of CD45 expression by MM cells. However, recent data indicate that VEGF plasma cell expression is similar between MGUS and MM patients suggesting that other molecules could be involved. In line with this hypothesis we have recently demonstrated that myeloma cells may also produce factors with angiogenic properties as angiopoietin-1 (ANG-1) and osteopontin (OPN) that are involved in myeloma induced angiogenesis in vitro. In order to identify which factors correlate with BM angiogenesis in MM patients, we have investigated in a cohort of 121 newly diagnosed MM patients (stage I–III) the expression of the angiogenic molecules VEGF, ANG-1 and OPN and their correlation with bone marrow (BM) angiogenesis and CD45 expression by MM cells. We found that 90% of CD138+ MM cells tested were positive for VEGF mRNA. On the other hand we found that 50% and 40 % of MM patients were positive for ANG-1 and OPN mRNA respectively. Using the previously published cut off for CD45 expression we found that 61 out of 121 MM patients were positive for CD45 and 60 out of 121 were negative for CD45 expression. Any correlation was not observed between VEGF expression and BM angiogenesis in MM patients (p=0.5), whereas the number of microvessels X field was higher in Ang-1 positive patients in comparison with Ang-1 negative ones (mean±SE: 6.23±0.2 vs. 2.94±0.1, median: 6.21 vs. 2.79; p=0.001,) and the microvascular density (MVD) was significantly increased (32.98±1.7 vs. 14.55±1.3, median: 34.69 vs. 13.04; p<0.01; capillaries: 26.73±1.3 vs. 10.42±0.8, median: 24.06 vs. 9.04; p<0.01, small venules: 9.56 ±0.5 vs. 4.14±0.5, median: 10.60 vs. 3.65; p<0.01). Furthermore a significantly positive correlation between Ang-1 expression and MVD was found (Pearson Chi-square: p=0.036, Cochran’s Linear Trend: p=0.01). A significantly higher MVD was also observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p<0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (mean±SE: 6.7±0.15 vs. 4.28±0.04; p=0.05). On the other hand, any significant difference was not observed between CD45 positive and CD45 negative patients for the expression of VEGF (p=0.4), ANG-1 (p=0.3) and OPN (p=0.09). Consistently we did not find any significant difference in both MVD and number of vessels X field between CD45 positive patients as compared with CD45 negative ones (p=0.5 and p=0.4, respectively). Finally, a multivariate analysis confirmed that VEGF and CD45 did not correlate with the BM angiogenesis showing that ANG-1 expression by MM cells was more tightly correlated with MVD and the number of vessels X field as compared to OPN. Our data indicate that ANG-1 and in part OPN rather than VEGF and CD45 expression by MM cells are the critical determinants correlated with the increase of BM angiogenesis that occurs in MM patients at the diagnosis.

A gp130 Interleukin-6 Transducer-Dependent SCID Model of Human Multiple Myeloma

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4727-4737 ◽  
Author(s):  
Cosette Rebouissou ◽  
John Wijdenes ◽  
Patrick Autissier ◽  
Karin Tarte ◽  
Valerie Costes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Scid Mice ◽  
Term Survival ◽  
Myeloma Cells ◽  
Tumoral Cells ◽  
Human Myeloma Cells

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)–related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6–dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

Hypoxia Activated Prodrug TH-302 for the Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3838-3838
Author(s):  
Jinsong Hu ◽  
Damian R Handisides ◽  
Els Van Valckenborgh ◽  
Hendrik De Raeve ◽  
Eline Menu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Solid Tumors ◽  
Cycle Phase ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Hypoxic Conditions

Abstract Abstract 3838 Poster Board III-774 Hypoxia is known to be linked to increased metastatic potential and a treatment-resistant phenotype leading to rapid progression and poor prognosis in solid tumors. We confirmed previous data[1] on hypoxia in human multiple myeloma (MM) in the 5T33MM syngeneic murine model of MM by using H&E staining and Hypoxyprobe (Pimonidazole) staining on consecutive serial sections from both naive mice and 5T33MMvv diseased mice. We observed a physiological hypoxic situation in MM diseased bone marrow. Given the contribution of hypoxia to tumor progression and drug resistance, a number of hypoxia-targeted therapeutics are under development. TH-302 is a new hypoxia-activated prodrug (HAP) that is currently being evaluated in the clinical trials as monotherapy and in combination with standard chemotherapy regimens for the treatment of solid tumors. The aims of the current study are (1) to demonstrate the effects of TH302 on MM cells in hypoxic conditions, focusing on apoptosis and cell cycle and associated signaling pathways and (2) to evaluate potential therapeutic effects when used in an experimental mouse MM model. We evaluated the effects of TH-302 in vitro on the murine 5T33MMvt cell line and the human LP-1, MMS-1, RPMI-8226, Karpas MM cell lines. Flow cytometry analysis revealed that TH-302 (0.5-50μM) can induce significant Go/G1 cell cycle phase arrest and apoptosis in hypoxic conditions (both 1% and 0% O2) in a concentration dependent manner, in contrast to normoxic conditions (20% O2) (p<0.001). Western blot confirmed that treatment with TH-302 in hypoxic conditions down-regulates cyclin D1/2/3, CDK4/6 and pRb expressions, but CDK2 expression was not disturbed. Furthermore, treatment with TH-302 in hypoxic conditions down-regulates the anti-apoptotic proteins BCL-2 and BCL-xL, as well as up-regulates the expression of three proapoptotic proteins: cleaved caspase-3, 9 and PARP. The expression pattern of Bax was however not influenced. The expression of p21 and p27 decreased in hypoxic condition after treatment with TH-302. Further studies conducted in the 5T33MMvv mouse model demonstrated that animals treated prophylactically with TH-302 (12.5 mg/kg, 25 mg/kg and 50 mg/kg, i.p.) for 3 weeks from day 1 after tumor inoculation showed decreased serum paraprotein (12.5 mg/kg, 32% decrease, p<0.05; 25 mg/kg, 77% decrease, p<0.001; 50 mg/kg, 54% decrease, p<0.001), compared to vehicle-treated 5T33MMvv mice (n=10). The frequency of apoptotic multiple myeloma cells in bone marrow sections was also significantly increased (12.5 mg/kg, 2.5 fold, p<0.05; 25mg/kg, 2.1 fold, p<0.05; 50mg/kg, 3.1 fold, p<0.01). Treatment with TH-302 resulted in no adverse events, any observable detriment to the mice or weight loss (p>0.05). In conclusion, these results show that hypoxia-activated treatment with TH-302 activates apoptosis and induces cell cycle arrest in MM cells, under hypoxic conditions, both in vitro and in vivo and therefore represents a promising therapeutic approach for multiple myeloma. Reference [1] Simona Colla, Paola Storti, Gaetano Donofrio, et al. Hypoxia and Hypoxia Inducible Factor (HIF)-1α in Multiple Myeloma: Effect on the Pro-Angiogenic Signature of Myeloma Cells and the Bone Marrow Microenvironment, 50th ASH annual meeting, http://ash.confex.com/ash/2008/webprogram/Paper13156.html Disclosures: Handisides: Treshold Pharmaceuticals: Employment. Liu:Treshold Pharmaceuticals: Employment. Sun:Treshold Pharmaceuticals: Employment. Hart:Treshold Pharmaceuticals: Employment. Vanderkerken:Treshold Pharmaceuticals: Research Funding.

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